Genetically modified microorganisms have recently attracted much interest as biofactories for production of foods, bioactive compounds, and biofuels. Spread of the genetically modified microorganisms outside of the intended place of cultivation into natural ecosystems, however, is a major regulatory concern. Some biological containment strategies can result in genetically modified microorganisms self-destructing by expression of heterologous genes encoding lethal proteins. Several bacterial toxins have been considered as good candidates for use in bacterial containment systems, including membrane-destabilizing or pore-forming proteins and enzymes attacking the genetic material of the cell. In many cases, however, mutation of toxin genes introduced into microorganisms results in reduced efficacy of toxin genes over time.
Type II toxin-antitoxin systems are widespread in prokaryotes (Van Melderen and De Bast (2009) PLoS Genetics 5: e1000437; Marakova et al (2009) Biology Direct 4:19). These toxin genes typically encodes proteins that interfere with transcription (e.g., by inhibiting DNA gyrase) or translation by interfering with ribosome function or by degrading RNA transcripts. Toxins with endoribonucleolytic activity are sometimes referred to as “RNA interferases” and include, for example, the bacterial toxins MazF, pemK, RelE, HicB, HipA, Doc, VapC, yafQ, yhaV, and tasB, among others. Expression of the toxin genes is tightly controlled by antitoxin genes which reside in an operon with the toxin gene. Typically the antitoxin is the first gene of the transcript and overlaps the toxin gene by 1-10 nucleotides, allowing for the antitoxin to be more efficiently translated with respect to the toxin. The antitoxin forms a stable complex with the toxin, resulting in inactivation of the toxin. The antitoxin-toxin complex also binds to the promoter of the TAS, repressing transcription. Thus, under ordinary circumstances, expression of the TAS is shut down—the antitoxin, which is produced in greater abundance, binds to and inactivates the toxin, and prevents further transcription of the toxin operon. The antitoxin protein is labile when not associated with the toxin however, and if the system becomes unbalanced, for example, by increased turnover of the antitoxin, the toxin can persist in the cell free of the antitoxin, where its endoribonucleolytic activity (in cases where the toxin is an “RNA interferase”) is able to shut down translation.
The elaborate mechanisms used to limit toxin expression in endogenous systems (see for example Diago Navarro et al. (2009) FEBS J. 277: 3097-3117 for a thorough treatment or the parDE TAS regulation) underscore the importance of tightly controlling the expression of an exogenous toxin gene introduced into the cell. The selective pressure to mutate the toxin to an inactive form has limited the potential of toxin genes in biocontainment. Further, in recent years several groups have suggested that native TAS may serve to promote cytostasis rather than cell growth under growth-limiting conditions, and that in many cases at least a portion of a population in which a toxin is activated subsequently recover (see, for example, Cataudella et al. (2012) Nucl Acids Res). The ability of cells to survive the expression of an active toxin has also been seen when exogenous genes were expressed in heterologous systems (e.g, Kristofferesen et al (2000) Appl Environ Microbiol 66: 5524-5526), also raising doubts about the practicality of using Type II TASs in biocontainment strategies.
Microorganisms make various metabolic adjustments in response to nutrient depletion, including, for example, transcriptional responses that allow increased uptake of external sources of nutrients as well as scavenging of internal sources. Much of the response to nutrient stress is based on transcriptional regulation of transporters, enzymes, proteins of the translational machinery, etc. Photosynthetic microorganisms that rely on light for chemical energy and carbon fixation, have additional challenges in that the photosynthetic apparatus must be adjusted to prevent excessive light damage to the cell when it may not be possible to maintain photosynthetic electron transport or carbon fixation at optimal levels. The inability to adjust light harvesting and photosystem function can lead to sustained damage to these systems. Not surprisingly, many studies have found that alterations of the photosynthetic apparatus are among the changes seen in transcriptional response to nitrogen (Miller et al (2010) Plant Physiol 154: 1737-1752) phosphate (Yehudai-Resheff et al, (2007) The Plant Cell 19: 1023-1038; Wurch et al (2011) Environ Microbiol 13: 468-481), sulfur (Moseley et al (2009) Genetics 181: 889-905)), iron (Merchant et al. (2006) Biochim Bioophys Acta 1763: 578-594), copper (Castruita et al. (2011) The Plant Cell 23: 1273-1292), and CO2 (Wang et al (2011) Phototsynth Res 109: 115-122) limitation in microalgae and have found that the inability to adjust to nutrient limitation results in death of microalgal cultures (Moseley et al. (2006) Eukaryot. Cell 5: 26-44).